Skip to main content
. 2019 Aug 21;87(9):e00457-19. doi: 10.1128/IAI.00457-19

FIG 5.

FIG 5

Viability and caspase-3/7 activation in U937 cells following infection with S. maltophilia wild-type and virB10 mutant strains. (A) Macrophage monolayers were left uninfected (UI) or infected with either parental strain K279a (WT), virB10 mutant NUS15 (virB10), or complemented mutant NUS15(pvirB10) (virB10/virB10+), and at 24 h postinoculation, the amount of viable host cells was enumerated. The raw data appear in the left panel, and data normalized to the value for uninfected cells are in the right panel. (B) Cell lysates were collected and analyzed by SDS-PAGE and then immunoblotted using an anti-caspase-7 antibody. The left panel depicts the immunoblot from a representative experiment, with the migration of molecular mass standards (in kDa) indicated to the left of the gel image and the migration of caspase-7 and its active 20- and 10-kDa cleaved products to the right of the gel image. Immunoblot analysis using anti-GAPDH antibodies was performed to confirm equal loading. The graph in the right panel shows the levels of the active cleaved products relative to levels for GAPDH pooled from three independent experiments. Asterisks indicate significant differences between strains (*, P < 0.05). (C) U937 macrophages were infected with the strains indicated in panel A, and at 24 h postinoculation, levels of caspase-3/7 cleavage were determined by CellEvent caspase-3/7 green detection reagent. UI cells were included as a negative control. The data are normalized to the value for cells infected with the WT, which was set at 100% activation. (D) After the addition of U937 cells into the lower chamber of a transwell apparatus, the bacterial strains indicated in panel C were added to the upper chamber of the transwell apparatus with a filter preventing contact between the bacteria and the host cells, and at 24 h postinoculation, the levels of caspase-3/7 cleavage were determined and presented as described for panel C. In panels A, C, and D, asterisks indicate significant differences between the strains and/or relative to the UI control (*, P < 0.05; **, P < 0.01; ***, P < 0.001), and data are presented as the means and standard deviations of results from three independent experiments (n = 3 each).